Project Summary: The long-term, broad objective of this project is to understand the intracellular, molecular mechanisms by which mammalian orthoreoviruses (reoviruses), which are known causes of human infections and established models for studies of viral pathogenesis, mediate the complex, dynamic processes of viral genome replication, packaging, and particle assembly. The specific aims of this proposal are focused on the viral non-structural protein muNS, which has been shown to play key roles in forming specialized cytoplasmic structures, the "viral factories", in which many of the steps in reovirus replication and assembly occur. Related structures are used for similar purposes by other members of the same taxonomic family of multi-segmented double-stranded RNA viruses, such as the human pathogen rotavirus and the livestock pathogen orbivirus. Proposed experiments will attempt to characterize features of the muNS protein that determine its capacity for self-association and association with cellular factors to induce the formation of factory-like inclusions in the absence of other reovirus proteins (Aim 2), as well as its capacity for association with other viral proteins and potentially RNAs as well to recruit them to the factories or factory-like inclusions (Aim 1). Other proposed experiments will attempt to refine and apply genetic approaches, including RNA interference and vector-based trans-complementation, for testing the roles of muNS and the factories in productive infections by reoviruses (Aim 3). Within the framework of these aims, other viral and cellular components are proposed to be studied insofar as they associate with muNS, with a special emphasis on the viral sigmaNS and mu2 proteins, which have also been implicated in determining features or functions of the factories. Relevance to public health: The intracellular steps in viral genome replication and particle assembly are still poorly understood for many viruses and are expected, once better understood, to present many new targets for anti-viral therapies. The reovirus factories provide a useful model in this regard. The particular virus on which this proposal focuses, mammalian reovirus, is the prototype of a taxonomic family that includes important pathogens of humans, food animals, and food plants, and is moreover under study as an oncolytic agent.